Common variants in the CLDN2-MORC4 and PRSS1-PRSS2 loci confer susceptibility to acute pancreatitis
Frank Ulrich Weiss
a,1, Nico Hesselbarth
b,1, Andrea P arniczky
c,d, Dora Mosztbacher
e, Felix L€ ammerhirt
a, Claudia Ruffert
b, Peter Kovacs
f, Sebastian Beer
g, Katharina Seltsam
g, Heidi Griesmann
b, Richard B€ ohme
b, Tom Kaune
b, Marcus Hollenbach
g,
Hans-Ulrich Schulz
h, Peter Simon
a, Julia Mayerle
a,i, Markus M. Lerch
a, Giulia Martina Cavestro
j, Raffaella Alessia Zuppardo
j, Milena Di Leo
j,
Pier Alberto Testoni
j, Ewa Malecka-Panas
k, Anita Gasirowska
k, Stanislaw G ł uszek
l, Peter Bugert
m, Andrea Szentesi
d,n, Joachim M€ ossner
g, Heiko Witt
o, Patrick Michl
b, Peter H egyi
d,p, Markus Scholz
q,r,1, Jonas Rosendahl
b,*,1aDepartment of Internal Medicine A, Ernst-Moritz-Arndt University, Greifswald, Germany
bDepartment of Internal Medicine I, Martin Luther University, Halle, Germany
cHeim Pal Children's Hospital, Budapest, Hungary
dInstitute for Translational Medicine and First Department of Internal Medicine, University of Pecs, Pecs, Hungary
eFirst Department of Pediatrics, Semmelweis University, Budapest, Hungary
fLeipzig University Medical Center, IFB Adiposity Diseases, University of Leipzig, Leipzig, Germany
gDepartment of Internal Medicine, Neurology and Dermatology, Division of Gastroenterology, University of Leipzig, Leipzig, Germany
hDepartment of Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
iDepartment of Medicine II, University Hospital, Ludwig-Maximilians-University Munich, Germany
jGastroenterology and Gastrointestinal Endoscopy Unit, Division of Experimental Oncology, Vita-Salute San Raffaele University, IRCCS Ospedale San Raffaele Scientific Institute, Milan, Italy
kDepartment of Digestive Tract Diseases, Medical University ofŁodz,Łodz, Poland
lFaculty of Medicine and Health Sciences, Jan Kochanowski University, Kielce, Poland
mInstitute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service of Baden- Württemberg, Mannheim, Germany
nFirst Department of Medicine, University of Szeged, Hungary
oElse Kr€oner-Fresenius-Zentrum für Ern€ahrungsmedizin (EKFZ), Paediatric Nutritional Medicine, Technische Universit€at München (TUM), Freising, Germany
pHAS-SZTE, Momentum Gastroenterology Multidisciplinary Research Group, Szeged, Hungary
qLIFE- Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany
rInstitute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany
a r t i c l e i n f o
Article history:
Received 6 April 2018 Received in revised form 29 May 2018
Accepted 31 May 2018 Available online 1 June 2018
Keywords:
Acute pancreatitis Genetics
a b s t r a c t
Background/Objectives: Acute pancreatitis (AP) is one of the most common gastrointestinal disorders often requiring hospitalization. Frequent aetiologies are gallstones and alcohol abuse. In contrast to chronic pancreatitis (CP) few robust genetic associations have been described. Here we analysed whether common variants in theCLDN2-MORC4and thePRSS1-PRSS2locus that increase recurrent AP and CP risk associate with AP.
Methods: We screened 1462 AP patients and 3999 controls with melting curve analysis for SNPs rs10273639(PRSS1-PRSS2),rs7057398(RIPPLY), andrs12688220(MORC4). Calculations were performed for the overall group, aetiology, and gender sub-groups. To examine genotype-phenotype relationships we performed several meta-analyses.
Abbreviations:ACP, alcoholic chronic pancreatitis; AP, acute pancreatitis; CI, confidence interval; CLDN2, claudin 2; CP, chronic pancreatitis; CTRC, chymotrypsin C; GWAS, genome wide association study; MORC4, MORC family CW-type zincfinger 4; NACP, non-alcoholic chronic pancreatitis; OR, odds ratio; PRSS1, serine protease 1, cationic trypsinogen; PRSS2, serine protease 2, anionic trypsinogen; RIPPLY1, ripply transcriptional repressor 1; SNP, single nucleotide polymorphism.
*Corresponding author. University Clinic and Policlinic of Internal Medicine I, University Clinic of Halle (Saale), Ernst-Grube Straße 40, 06120, Halle, Germany.
E-mail address:jonas.rosendahl@uk-halle.de(J. Rosendahl).
1 These authors contributed equally to this work.
Contents lists available atScienceDirect
Pancreatology
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p a n
https://doi.org/10.1016/j.pan.2018.05.486
1424-3903/©2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.
Single nucleotide polymorphisms
Risk factors Results:Meta-analyses of all AP patients depicted significant (p-value<0.05) associations forrs10273639 (odds ratio (OR) 0.88, 95% confidence interval (CI) 0.81e0.97,p-value 0.01),rs7057398(OR 1.27, 95% CI 1.07e1.5,p-value 0.005), andrs12688220(OR 1.32, 95% CI 1.12e1.56,p-value 0.001). For the different aetiology groups a significant association was shown forrs10273639(OR 0.76, 95% CI 0.63e0.92,p-value 0.005),rs7057398(OR 1.43, 95% CI 1.07e1.92,p-value 0.02), andrs12688220(OR 1.44, 95% CI 1.07e1.93, p-value 0.02) in the alcoholic sub-group only.
Conclusions: The association of CP risk variants with different AP aetiologies, which is strongest in the alcoholic AP group, might implicate common pathomechanisms most likely between alcoholic AP and CP.
©2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.
Introduction
Acute pancreatitis (AP) is one of the most common gastroin- testinal disorders requiring hospital admission worldwide [1].
Accordingly, treatment costs are a major burden for health care systems as demonstrated for the United States of America [2,3]. The course of the disease varies and is more severe when necrosis de- velops while it is worst in patients with infected necrosis. So far, treatment options are symptomatic since causal interventions are not available.
In a large proportion of patients, alcohol misuse or bile duct stones cause AP, whereas other aetiologies are rare [4,5]. In contrast, to chronic pancreatitis (CP) where several genetic risk factors have been described for alcoholic (ACP) as well as the non- alcoholic form (NACP) there are only a few replicated genetic as- sociations with common variants found in AP. Here, an association with the p.N34S variant in theSPINK1gene (serine protease Kazal, Type 1) was found in a large European cohort as well as an asso- ciation with the c.180C>T variant (p.G60¼) inCTRC(Chymotryp- sinogen C) [6,7]. Other studies investigating common variants in cytokines, barrier function, and the innate and adaptive immune system did not reveal robust associations [8,9]. Recently, a genome- wide association study (GWAS) in patients with chronic and recurring pancreatitis detected variants in the CLDN2-MORC4 (claudin 2; RIPPLY1, ripply transcriptional repressor 1; MORC family CW-type zincfinger 4) andPRSS1-PRSS2(serine protease 1 and 2, cationic and anionic trypsinogen) loci that were associated with the disease [10]. The results werefirst replicated in a European cohort and thereafter in Asian cohorts as well [11,12]. Interestingly, the association seems to be strongest in ACP patients, a result which was confirmed in a large European GWAS [13]. As recent studies showed that ACP and NACP most likely share mutual patho- mechanisms [11,13], we aimed to investigate whether genetic var- iants in the reported common risk loci also associate with AP. This hypothesis was further supported by an Indian study in patients with recurring idiopathic AP that demonstrated an association of theCLDN2-MORC4and thePRSS1-PRSS2variants in this cohort [14].
Here, we present the results of a large-scale association analysis of these variants in an European cohort including 1462 patients with AP of different aetiologies and 3999 healthy controls.
Methods Study subjects
The respective medical ethical review committees of all participating centres approved the study protocol and all patients gave written informed consent. The diagnosis of AP was made when at least two of the following criteria were fulfilled: a) typical clinical presentation (upper abdominal pain); b) elevation of serum lipase or amylase levels 3-times above the upper limit; c) imaging
studies indicating AP [15]. The aetiology of AP was categorized into alcoholic, biliary, idiopathic, trauma, post-ERCP, hyper- triglyceridaemia, hypercalcaemia, medication and drugs, ischemic, anatomic variants, and unknown. Given the limited sample size in these categories, sub-group analyses were performed for the alcoholic, biliary, and idiopathic cohorts only. Further follow-up data of the patients were not available. In total, the study included 1462 AP patients (n¼672 women), derived from Ger- many (n¼777; 339 women), Italy (n¼164; 86 women), Hungary (n¼501; 245 women), and Poland (n¼20; 2 women). When the patients were stratified according to the revised Atlanta classifica- tion the following distribution was observed for the overall AP group with available data (n¼1300), mild cases n¼761; moder- ately severe cases n¼363; severe cases n¼176 [16]. The detailed distribution of patients and controls is summarized inTable 1.
As controls we used in parts formerly genotyped samples from our recent study [11]. Overall, 3999 samples were used (2220 women) from Germany (n¼3295; 1828 women), Italy (n¼326;
221 women), Hungary (n¼246; 138 women), and Poland (n¼132;
63 women). More details of the controls are provided in Supplementary Table 1.
Genotyping
The detailed methods used for genotyping are summarized in the Online Supporting Information. For quality control we re- genotyped three percent of the samples blinded to the investi- gator. The concordance rate was 97.4% (453/465). Call rates for rs10273639,rs7057398andrs12688220 in the European samples were 99.3% (5421/5461), 98.6% (5383/5461), and 99.3% (5425/
5461), respectively.
Statistical analysis
The methodology used for the statistical analyses has been recently described elsewhere [11]. In brief, we assessed the quality of SNP genotypes by study-wise call rate. Hardy-Weinberg equi- librium was calculated for all patients and controls in thePRSS1- PRSS2 and for females in the CLDN2-MORC4 locus. SNPs in the CLDN2-MORC4locus were analysed separately for female and male patients. Effects of the SNPs were determined by logistic regression analysis using an additive model of inheritance. We used a model of complete X (XIA) and no X inactivation (nXIA) to determine com- bined effects [16]. Regarding analysis of X-chromosomal SNPs, male genotypes A and B are always coded as 0 and 1, respectively. In the model of X inactivation, female genotypes AA, AB, BB are coded as 0, 0.5, 1, while in the model of no X inactivation, these genotypes are coded as 0, 1, 2. Overall statistics and stratified tests for Hardy- Weinberg disequilibrium were additionally performed [17]. We pooled study-wise effects by standard meta-analysis techniques as implemented in the package“meta”of the statistical software“R
3.0.1” (ww.r-project.org). Heterogeneity between studies was assessed using Q-statistics. As we occasionally observed study heterogeneity, we calculated random-effect models throughout.
We analysed and compared likelihoods of XIA, nXIA and sex interaction for the purpose of model diagnostics. We generated forest-plots using GraphPad Prism (v.6.0a) (San Diego).P-values
<0.05 were considered statistically significant.
Results
PRSS1ePRSS2 locus (rs10273639)
Our meta-analysis of all AP patients in contrast with all controls revealed a significant association (odds ratio (OR) 0.88, 95% confi- dence interval (CI) 0.81e0.97,p-value 0.01) for the PRSS1-PRSS2 locus. In the country-specific subgroups this association was sig- nificant in Hungarian (OR 0.73, 95% CI 0.59e0.91,p-value 0.0043) and German patients (OR 0.89, 95% CI 0.79e0.99,p-value 0.044) (Fig. 1). Contrary to male patients (OR 0.83, 95% CI 0.73e0.94,p- value 0.004), no significance was observed for the female group of all AP patients (Supplementary Table 2). Next, we analysed the different aetiology subgroups of all patients and detected a signif- icant association for the alcoholic AP subgroup (OR 0.76, 95% CI
0.63e0.92,p-value 0.005). This association was also detected in the total male alcoholic AP patients (OR 0.77, 95% CI 0.62e0.95,p-value 0.015), but not in the female patients. All other analysis including comparisons of subgroups in females and males as well as between the different aetiology groups were not significant. The results are summarized inFig. 1,Supplementary Fig. 1 AeCandSupplementary Table 2. Genotype frequencies are shown inSupplementary Table 5.
RIPPLY1 (rs7057398)
Using the model with X-inactivation the meta-analysis of all AP patients compared with all controls revealed a significant associa- tion (OR 1.27, 95% CI 1.07e1.5,p-value 0.005) forRIPPLY1in the CLDN2-MORC4locus. In the country specific subgroups from Italy and Poland this association was statistically significant (OR 1.83, 95% CI 1.07e3.15, p-value 0.028; OR 6.48, 95% CI 2.24e19.55, p- value 0.0006; respectively). Fig. 2summarizes the results for all patients and the country-specific subgroups. In the gender specific subgroups a statistically significant association was seen for all male patients (OR 1.25, 95% CI 1.02e1.53,p-value 0.028) and the male patients from Poland (OR 6.59, 95% CI 2.16e21.2, p-value 0.001). In the analysis of the aetiology subgroups a significant as- sociation was seen for the overall alcoholic group (OR 1.43, 95% CI Table 1
Details of the patient and control cohorts.
Country Germany Hungary Italy Poland All
Patients
Age range years 9e96 11e93 18e87 24e92 9e96
Median 52 58 56 48 54
No. (female) 777 (339) 501 (245) 164 (86) 20 (2) 1462 (672)
Aetiology
Biliary 315 219 38 8 580
Alcohol 176 80 10 11 277
Idiopathic 168 84 17 0 269
Unknown 43 66 91 0 200
Post-ERCP 33 19 3 1 56
HTG, HCa 13 29 0 0 42
Drugs and medication 15 3 1 0 19
Trauma 12 0 0 0 12
Anatomic variants 2 0 4 0 6
Ischemia 0 1 0 0 1
Controls
Age range years 18e81 18e89 18e83 16e91 16e91
Median 52 45 35 34 49
No. (female) 3295 (1828) 246 (108) 326 (221) 132 (63) 3999 (2221)
Genotyped in Derikx et al. (2015) 2853 35 326 89 3303
The controls were in parts genotyped previously with the same methodology (Derikx et al., Gut, 2015). Abbreviations: ERCP, endoscopic retrograde cholangiopancreatog- raphy; HTG, hypertriglyceriaemia; HCa, hypercalcaemia; No., number.
Fig. 1.Results forrs10273639(PRSS1ePRSS2) for the overall acute pancreatitis cohorts from the different countries and corresponding Meta-analysis results. Separated by the dashed lines, Meta-analysis results of the different aetiologies (biliary¼bili, alco- holic¼alc, and idiopathic¼idi) are shown. Numbers of patients and controls (co.) are given in parentheses. To summarize the single study results, afixed-effect model was used.
Fig. 2.Results forrs7057398(RIPPLY1) for the overall acute pancreatitis cohorts from the different countries and corresponding Meta-analysis results. Separated by the dashed lines, Meta-analysis results of the different aetiologies (biliary¼bili, alco- holic¼alc, and idiopathic¼idi) are shown. Numbers of patients and controls (co.) are given in parentheses. Afixed effect meta-analysis assuming complete X-inactivation was performed.
1.07e1.92,p-value 0.02), only. The results are summarized inFig. 2, Supplementary Fig. 2 A-Cand in detail including all aetiology as well as gender specific subgroups inSupplementary Table 3 A. The associations under the non X-inactivation model were comparable (Supplementary Table 3 B). The genotype (for females) and allele frequencies (for males) are shown inSupplementary Tables 6 and 7.
MORC4 (rs12688220)
Furthermore, for the model of X-inactivation a statistically sig- nificant association was found for the group of all AP patients (OR 1.32, 95% CI 1.12e1.56,p-value 0.001) investigating MORC4. A sig- nificant association was also seen in the country specific subgroups from Italy (OR 2.1, 95% CI 1.2e3.69,p-value 0.0094) and Poland (OR 5.9, 95% CI 2.05e17.63,p-value 0.001).Fig. 3summarizes the results for all patients and the country-specific subgroups. In the aetiology subgroups, association with all alcoholic AP patients (OR 1.44, 95%
CI 1.07e1.93, p-value 0.02) was found only. In gender specific subgroups no significant associations were present, apart from the overall AP male patient group (OR 1.33, 95% CI 1.08e1.63,p-value 0.006) and the male alcoholic AP group (OR 1.47, 95% CI 1.07e2.01, p-value 0.02). The results are summarized inSupplementary Fig. 3 A-Cand in detail including all aetiology as well as gender specific subgroups inSupplementary Table 4 A. The associations in the non X-inactivation calculations were similar (Supplementary Table 4 B).
The genotype (for females) and allele frequencies (for males) are shown inSupplementary Tables 8 and 9.
Analysis of aetiologies and disease course
The strength of the associations differed numerically between the aetiology subgroups. However, when subgroups were analysed no significance was obtained (data not shown). Additionally, we compared the severity sub-groups categorized with the revised Atlanta classification (mild, moderately severe, severe) [18], but did not detect a significant association (data not shown).
Discussion
The main mechanism for the pathogenesis of CP seems to depend on premature intra-pancreatic trypsin activation or its diminished inhibition. This pathophysiological concept is sup- ported as several genetic associations have been described in genes encoding proteases or anti-proteases of the digestive enzyme cascade [19]. For thefirst time our results demonstrate an associ- ation of a common protective variant in thePRSS1-PRSS2locus with AP. Functional studies of this variant indicate a lower intra- pancreatic trypsinogen level, which would fit in the proposed concept [20]. However, the strength of the association in AP (all
patients, OR 0.88, 95% CI 0.81e0.97) is lower compared to thefirst report in patients with CP or recurrent AP (OR range from 0.71- 0.748) or to European ACP patients (OR 0.63, 95% CI 0.55e0.72) [10,11]. Of note, the effect size of the SNPs was strongest in the alcoholic AP group. As such, one might argue that our observed association is explained by the fact that alcoholic AP progresses to ACP in up to 38% of the patients [21]. When we compared contrasts of the associations between aetiology groups no significant differ- ences were obtained indicating that our observed association is not driven by the alcoholic AP sub-group. As patient numbers in the subgroups were small we, however, might have missed the effect in this comparison. Nevertheless, it is conceivable that the trypsin- mediated pathomechanism is also relevant for the development of AP in patients with different aetiologies. Here, the disturbed balance of proteases and anti-proteases seems to represent the initiating event in pancreatitis development, whereas the mecha- nisms important for the disease course or the development of CP still need to be elucidated.
Apart from the trypsin-mediated pathway associations of vari- ants in the X-chromosomal CLDN2-MORC4 locus with AP were found. The strength of the association for the overall cohorts was comparable for the RIPPLY SNP (rs7057398) and the MORC4 (rs12688220) variant (OR 1.27, 95% CI 1.07e1.5; OR 1.32, 95% CI 1.12e1.56). Compared to the initial GWAS (1.21e1.49) the associa- tion ofrs7057398was similar, whereas it was slightly stronger in female European ACP patients (OR 1.57) and male ACP patients (OR 2.26) formerly reported [10,11]. Again, for theMORC4 rs12688220 variant the association (OR 1.33) was comparable to that reported in the GWAS (1.24e1.61), but weaker than in European ACP patients (female, OR 1.71; male, 2.66).
In our study, no significant differences in female and male subgroups were found and models with or without X-inactivation showed no differences. This implies that the variants do not have a gender specific effect. As mechanism of action of theCLDN2-MORC4 risk variants, changes in the localization of claudin-2 were dis- cussed, which would represent a new disease causing mechanism [10]. Nevertheless, further functional studies are warranted to better understand the contribution of these variants to pancreatitis development. Summarized, our findings demonstrate that addi- tional pathways seem to be important for AP development, as it is the case for CP.
As the disease course in AP can vary dramatically, we wondered whether the investigated SNPs might influence severity of the disease in our patients. When we compared the associations of the variants in patients with mild, moderately severe, and severe AP classified according to the revised Atlanta classification no such correlation was seen. Again, this result implicates that variants in both loci may trigger disease onset, but do not influence the disease course.
A limitation of our study is the small sample size in some of the aetiology sub-groups. This entails that in these cohorts interpre- tation of the data requires caution. On the other hand, associations in theCLDN2-MORC4locus revealed homogenous results indicating reliability of the results. Although the comparison of the fre- quencies of the three SNPs between the aetiology subgroups did not identify that the association is driven by the alcoholic subgroup only, the association is strongest in this group and might be a replication of the results in chronic and recurrent acute pancrea- titis. Additionally, we have no follow-up data of the patients with alcoholic AP that might have developed chronic pancreatitis in the meantime. Therefore the effect seen in our analysis might be influenced by such a subgroup.
In conclusion, our study for thefirst time demonstrates an as- sociation of common CP SNPs with AP. This observation shows that the acute and the chronic form of the disease might share common Fig. 3.Results forrs12688220(MORC4) for the overall acute pancreatitis cohorts from
the different countries and corresponding Meta-analysis results. Separated by the dashed lines, Meta-analysis results of the different aetiologies (biliary¼bili, alco- holic¼alc, and idiopathic¼idi) are shown. Numbers of patients and controls (co.) are given in parentheses. Afixed effect meta-analysis assuming complete X-inactivation was performed.
pathomechanisms. This motivates the search for therapeutic ap- proaches addressing common mechanisms rather than different aetiologies. However, it remains unclear, which genetic factors in- fluence the disease course as well as the progression to CP.
Competing interests
There are no competing interests to be stated by any of the authors.
Financial disclosures None.
Acknowledgments
The authors thank all study participants for providing clinical data and blood samples. We also thank Knut Krohn, Kathleen Sch€on and Birgit Oelzner (IZKF core unit DNA technologies, Leipzig) for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants RO 3929/1-1, RO 3929/2-1&RO3929/5-1 (to J.R.), Wi 2036/2-2&Wi 2036/2-3 (to H.W.), GRK1947, A3 to J.M. and by the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1501 (AD2-06E99 to P.K.), by a grant of the Colora Stiftung gGmbH (to J.R.), the Else Kr€oner-Fresenius-Foundation (EKFS) (to H.W.), by grants of the European Regional Development Fund V-630-F-150-2012/133 and V630-S-150-2012/132 (to F.U.W.). LIFE is funded by means of the European Union, by the European Regional Development Fund (ERDF) and by funds of the Free State of Saxony within the frame- work of the excellence initiative (project numbers 713-241202, 14505/2470, 14575/2470). This study was supported by Project Grants K116634 and KH125678 (to P.H.) and Economic Develop- ment and Innovation Operative Programme Grant GINOP 2.3.2-15- 2016-00048 (to P.H.) of NKFIH. Andrea Parniczky and Dora Mosztbacher were supported with generous fellowships by the European Pancreatic Club.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://doi.org/10.1016/j.pan.2018.05.486.
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